Many hemoglobinopathies including sickle cell anemia and beta-thalassemia arise from single genetic point mutations resulting in the production of aberrant mRNA producing alternate splicing and/or protein products. To address therapeutic approaches to these genetic diseases, we have focused on site-directed nucleotide exchange by gene targeting to the abnormal globin gene in hematopoietic cells, specifically stem cells. The strategy under current investigation is use of chimeric RNA/DNA oligonucleotides to mediate site-directed mutagenesis via triple helix formation. The chimeric oligonucleotide composed of DNA and RNA residues in a duplex conformation is designed to form a more stable triple helix compared with a DNA triple helix. The chimeric oligonucleotide is chosen to be relatively small in size (68nts) and such that the nucleotide targeted for exchange is in the center of the complex should a triple helix form. The RNA/DNA sequence is complementary to that of the target gene with the single nucleotide mismatch. It is hypothesized that the unpaired nucleotide will be recognized by endogenous repair system and result in the alteration of the DNA sequence of the targeted gene. A test plasmid has been constructed using a mammalian expression vector to drive expression of the mutated beta globin gene carrying the sickle cell anemia mutation and stable transformants of K562 cells have been generated. Transfer of the chimeric RNA/DNA oligonucleotides into hematopoietic K562 cells suggest that liposome mediated transfer is more appropriate in these studies than electroporation. A strategy was developed to detect specific gene targeting using PCR with radioactive phosphorous-labeled nucleotides and restriction enzyme digestion. Preliminary results suggest that the chimeric RNA/DNA oligonucleotide strategy may be successful for targeting of select globin gene mutations in hematopoietic cells, but at a very low level. Optimizing the condition for targeting and determining the mechanism of action of the chimeric RNA/DNA oligonucleotides is necessary to demonstrate the potential of this strategy for introducing or correcting gene mutations in situ.